DNA-binding motor proteins play critical roles in the promotion and regulation of homologous recombination (HR), and are indispensable for cancer avoidance in mammals. For instance, mutations in the motor proteins BLM and RECQ5 lead to aberrant recombination events and are associated with tumorigenesis, and mutations in Rad54 and Rad54B are found in a variety of tumor types. Understanding the properties of these motor proteins, and delineating their roles in genome maintenance, will be essential for revealing the molecular basis for defects that produce the disease phenotypes. The Rad54 protein is a key factor in homologous recombination and damage avoidance, and has been postulated to act at pre-synaptic, synaptic and post- synaptic steps in double strand break repair through homologous recombination. Despite this central role for Rad54 in the process, its exact function and regulation in vivo remain unknown, despite the wealth of biochemical studies characterizing its dsDNA-dependent ATPase activity and translocation on dsDNA. srs2 rad54 double mutants are lethal, due to action of Rad51 to promote HR, indicating an overlap in an essential step in HR. Our hypothesis is that the Rad54 DNA motor protein contributes to genome maintenance through a dynamic cycle of phosphorylation/dephosphorylation and this affects its ability to interact with Rad51 recombinase and function in HR. The essential step performed by Srs2 or Rad54 involves setting up the synaptic intermediate after a search for homology has been initiated. To test this hypothesis we will take a multidisciplinary approach that integrates our expertise in genetics (H. Klein) complemented by the biochemistry (P. Sung), and single-molecule biophysics (E. Greene) expertise of our collaborators, in order to conduct a detailed analysis of the S. cerevisiae Rad54 and Srs2 DNA motor proteins. Together, we are well positioned to decipher the mechanisms of these motor proteins and provide a valuable experimental framework for understanding the genome maintenance and tumor suppression roles of their mammalian counterparts.